Research in the laboratory is focused on the chemistry and biochemistry of nucleic acids with an emphasis on biologically important reactions involving RNA. Large RNAs and complex ribonucleoprotein machines such as the spliceosome and ribosome play a key role in constitutive and regulated cellular processes and in the life cycle of viral pathogens. There is thus a need for detailed structural and functional information regarding these molecules. A combination of chemical and biochemical approaches along with simple molecular biological manipulations provides a powerful approach for the analysis of such systems. Modified nucleic acid substrates, synthesized using both chemical and enzymatic methodologies, are very useful probes of the structure and reactions of nucleic acids and nucleic acid-protein complexes. Specific areas of interest include:
(1) Mechanisms of pre-mRNA Splicing
Intron excision from a pre-mRNA substrate involves two sequential transesterification reactions catalyzed by the spliceosome. These reactions are strikingly similar, mechanistically, to those of the self-splicing Group II introns although the evolutionary relationship between the two systems is unclear. It is widely assumed that the catalytic core of the spliceosome is an RNA structure; however, little is known about the chemical mechanisms of the transesterification reactions or the structure of the substrate bound at the active site. We are addressing these issues by structure/function studies involving chemically modified pre-mRNA splicing substrates. In addition, we are developing chemical footprinting reagents to explore the RNA structures at the heart of the splicing machinery.
(2) RNA Editing
RNA editing, involving the deamination of adenosine to inosine, is a recently characterized form of post-transcriptional RNA modification. This reaction alters codons in neural-expressed glutamate receptors and is also responsible for a codon change in the hepatitis delta virus RNA. We are studying this system with a focus on the basis of protein-RNA recognition and the nature of enzymatic catalysis within the context of a complex RNA molecule.
(3) RNA Folding and Dynamics
Large RNAs fold into complex structures which determine their activity and dynamic changes in RNA structure are required during many RNA processing events. We have developed methodologies to explore dynamic changes in RNA structure occuring with half-lives of several seconds or greater and are extending these types of studies to events on the millisecond time-scale. These time-resolved or "kinetic" footprinting tools have proved useful in examining the folding paths of the Group I RNA from Tetrahymena and M1 RNA the catalytic component of E. coli RNase P. Kinetic footprinitng should also provide valuable insight into dynamics which occur during the course of complex biochemical events such as pre-mRNA processing or translation.